Apoptosis of cardiac myocytes is one of the causes of heart failure. Here we examine the mechanism by which the activation of beta-adrenergic receptor induces cardiomyocyte apoptosis. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling and DNA ladder analyses revealed that isoproterenol (Iso) induced the apoptosis of cardiac myocytes of neonatal rats through an increase in intracellular Ca(2+) levels. The Iso-induced cardiomyocyte apoptosis was strongly inhibited by the L-type Ca(2+) channel antagonist nifedipine and by the calcineurin inhibitors cyclosporin A and FK506. Iso reduced the phosphorylation levels of the proapoptotic Bcl-2 family protein Bad and induced cytochrome c release from mitochondria to the cytosol through calcineurin activation. Infusion of Iso increased calcineurin activity by approximately 3-fold in the hearts of wild-type mice but not in the hearts of transgenic mice that overexpress dominant negative mutants of calcineurin. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling analysis revealed that infusion of Iso induced apoptosis of cardiac myocytes and that the number of apoptotic cardiomyocytes was significantly less in the hearts of the transgenic mice compared with the wild-type mice. These results suggest that calcineurin plays a critical role in Iso-induced apoptosis of cardiac myocytes, possibly through dephosphorylating Bad.